1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for standard, 1 well for blank.

Add 100μL each of dilutions of standard （read Reagent Preparation）, blank and samples into the appropriate

wells. Cover with the Plate sealer. Incubate for 2 hours at 37oC.

2. Remove the liquid of each well, don’t wash.

3. Add 100μL of working solution to each well. Incubate for 1 hour at 37oC after covering

it with the Plate sealer.

4. Aspirate the solution and wash with 350μL of 1× Wash Solution to each well using a squirt bottle,

multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Remove the remaining

liquid from all wells compley by snapping the plate onto absorbent paper. Totally wash 3 times. After the

last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against

absorbent paper.

5. Add 100μL of working solution to each well. Incubate for 30 minutes at 37oC after

covering it with the Plate sealer.

6. Repeat the aspiration/wash process for total 5 times as conducted in step 4.